Exposure of PC12 cells to ethanol results in large changes in calcium- stimulated protease activities. Since these proteases are critical modulators of both neurotransmitter release as well as cellular toxicity and death, we are exploring the hypothesis that ethanol- mediated neural toxicity may result from calcium-activated protease dysregulation. Calpastatin, an inhibitor of calpain, is an acidic, hydrophobic protein which interacts with the hydrophobic active site(s) of mu- and m-calpains. A series of post-translational modifications of calpastatin have been described which alter the binding affinity to calpains, among them, PKC-mediated phosphorylations. Using a PC12 model, we are examining the effects of ethanol exposure and withdrawal on protein-protein interactions. Because of the hydrophobic nature of calpastatin-calpain interactions, we examined the possibility that ethanol might modify protease-calpastatin (inhibitor) complex stability. We found that exposure of PC12 cells to ethanol results in an increase in the molecular weight of calpain and calpastatin- containing protein complexes, and that this is associated with a change in protease activity. We are extending these observations by use of immunocytochemical techniques, where the nature of these complexes and their cellular locations will be examined. - alcoholism, neurotoxicity, PC12, post-translational modifications, protein-protein interactions, calpain, calpastatin, proteases